1. Field of the Invention
This invention relates to hemoglobin receptor genes and the proteins encoded therefrom of certain bacterial species, particularly species of Neisseria bacteria. More particularly, this invention relates to hemoglobin receptor genes, polypeptides and peptides useful for preparing vaccines and antibodies against Neisseria, and methods and means for producing such peptides and polypeptides in vitro. Also provided are diagnostic and therapeutic methods and reagents useful in detecting and treating Neisseria infection and methods for developing novel and effective anti-Neisseria agents.
2. Background of the Invention
The Neisseriae comprise a genus of bacteria that includes two gram-negative species of pyogenic cocci pathogenic for humans: Neisseria meningitidis and Neisseria gonorrhoeae. N. meningitidis is a major cause of bacterial meningitis in humans, especially children. The disease characteristically proceeds from asymptomatic carriage of the bacterium in the nasopharynx to invasion of the bloodstream and cerebrospinal fluid in susceptible individuals.
N. meningitidis is one of the leading causes of bacterial meningitis in children and healthy adults in the world. The severity of the disease is evidenced by the ability of meningococci to cause the death of previously healthy individuals in less than 24 hours. N. meningitidis has a polysaccharide capsule whose diversity of component antigenic polysaccharide molecules has resulted in the classification of ten different serogroups. Of these, group A strains are the classic epidemic strains; group B and C are generally endemic strains, but C occasionally causes an epidemic outbreak. All known group A strains have the same protein antigens on their outer membranes, while group B strains have a dozen serotypes or groupings based on the presence of principal outer membrane protein antigens (as opposed to polysaccharides).
Survival of a pathogen such as N. meningitidis in a host depends on its ability to overcome a battery of host defense mechanisms. One nonspecific host defense mechanism against microbial intruders is to limit the availability of iron in tissues (Weinberg, 1984, Physiological. Rev. 64: 65-102), because iron is a necessary nutrient for most microbial pathogens. The vast majority of iron in the human adult is located intracellularly in the form of hemoglobin (76%) or ferritin (23%). The remainder can be found extracellularly bound to host iron-binding proteins such as transferrin and lactoferrin (Otto et al., 1992, Crit. Rev. Microbiol. 18: 217-233).
Pathogenic bacteria have adapted to this iron-limiting environment by developing highly specific and effective iron assimilation systems. A large number of these bacteria secrete siderophores, small, non-protein iron chelators which, due to their extremely high affinity for iron (III), scavenge trace amounts of iron(III) from the environment and shuttle the iron back to the bacterial cell (Baggs and Neilands, 1987, Microbiol. Rev. 51: 509-518; Braun and Hantke, 1991, in Winkelmann (ed.), Handbook of Microbial Iron Chelates, CRC Press: Boca Raton, Fla., pp. 107-138.).
Alternatively, some bacterial pathogens, like Neisseriae species (Archilbald and DeVoe, 1979, FEMS Microbiol. Lett. 6: 159-162; Mickelson et al., 1982, Infect. Immun. 35: 915-920; Dyer et al., 1987, Infect. Immun. 55: 2171-2175), Haemophilus influenzae (Coulton and Pang, 1983, Curr. Microbiol. 9: 93-98; Schryvers, 1988, Mol. Microbiol. 2: 467472; Jarosik et al., 1994, Infect. Immun. 62: 24702477), Vibrio cholerae (Stoebner and Payne, 1988, Infect. Immun. 56:2891-2895; Henderson and Payne, 1994, J. Bacteriol. 176: 3269-3277), Yersiniae (Stojiljkovic and Hantke, 1992, EMBO J. 11: 4359-4367) and Actinobacillus pleuropneumoniae (Gerlach et al., 1992, Infect. Immun. 60: 3253-3261) have evolved more sophisticated mechanisms to sequester iron from the host. These pathogens can directly bind host's iron-binding proteins such as lactoferrin, transferrin, and heme-containing compounds, and use them as sole sources of iron.
The importance of iron in the virulence of N. meningitidis was demonstrated by in vivo studies using mice as the animal model system (Calver et al., 1976, Can. J. Microbiol. 22: 832-838; Holbien et al., 1981, Infect. Immun. 34: 120-125). Specific iron-regulated outer membrane receptors have been shown to be involved in the binding and the utilization of lactoferrin- and transferrin-iron in Neisseriae (Schryvers and Morris, 1988, Infect. Immun. 56: 1144-1149 and Mol. Microbiol. 2:281-288; Legrain et al., 1993, Gene 130: 81-90; Pettersson et al., 1993, Infect. Immun. 61: 47244733 and 1994, J. Bacteriol. 176: 1764-1766). These receptors share significant amino acid similarity and, most probably, also the mechanism of iron internalization, with receptors for siderophores and vitamin B12 of other Gram-negative bacteria (Cornelissen et al., 1993, J. Bacteriol. 174: 5788-5797). In contrast, the mechanism by which Neisseriae utilize hemoglobin- and hemin-iron as well as the components involved have so far not been described.
Recently, several proteins with hemoglobin-binding and/or hemin-binding activities have been identified in total membranes of iron-limited N. meningitidis and N. gonorrhoeae.
Lee and Hill, 1992, J. gen. Microbiol. 138: 2647-2656 disclose the specific hemoglobin binding by isolated outer membranes of N. meningitidis.
Martek and Lee, 1994, Infect. Immun. 62: 700-703 disclosed that acquisition of heme iron by N. meningitidis does not involve meningococcal transferrin-binding proteins.
Lee, 1994, Microbiol. 140: 1473-1480 describes the biochemical isolation and characterization of hemin binding proteins from N. meningitidis.
The precise role of these proteins in hemin and/or hemoglobin utilization remains unclear at present, although these proteins are likely to be components of a hemin-utilization system in N. meningitidis.
The dependence on host iron stores for Neisseria growth is a potentially useful route towards the development of novel and effective therapeutic intervention strategies. Historically, infections of both N. meningitidis and N. gonorrhoeae were treated chemoprophylactically with sulfonamide drugs. However, with the development of sulfonamide-resistant strains came the necessity of using alternative modes of therapy such as antibiotic treatment. More recently, the drug treatment of choice includes the administration of high grade penicillin. However, the success of antimicrobial treatment is decreased if therapy is not initiated early after infection.
Gonococcal infection has also been treated with penicillin, ampicillin, or amoxicillin, tetracycline hydrochloride, and spectinomycin. Unfortunately, because the incidence of infections due to penicillinase-producing bacteria has increased, several new, more expensive .beta.-lactam antibiotics have been used in treatment. Despite the fact that existing antibiotics have decreased the serious consequences of gonorrhea, their use has not lowered the incidence of the infection in the general population.
Prevention of meningococcal disease has been attempted by chemoprophylaxis and immunoprophylaxis. At present, rifampin and minocycline are used, but only for humans in close contact with an infected person as this treatment has a number of disadvantages. The only commercially available vaccine against meningococcal meningitis has as its major component the bacterial polysaccharide capsule. In adults this vaccine protects against serogroups A, C, Y and W135. It is not effective against serogroup B, and is ineffective in children against serogroup C. Thus far, immunoprophylatic preventive treatment has not been available for N. gonorrhoeae.
Thus, what is needed are better preventative therapies for meningococcal meningitis and gonorrhea including more effective, longer lasting vaccines which protect across all of the serogroups of N. meningitidis and all the serotypes of N. gonorrhoeae. In addition, better methods are need to treat meningococcal and gonococcal infection.